Project Summary Chronic kidney disease (CKD) affects 9-14% of the U.S. adult population, and its progression is associated with a number of serious complications. Loss of functional nephrons contributes to progression of CKD, eventually leading to end-stage kidney disease (ESKD) where patients require kidney replacement therapies such as hemodialysis to survive. The treatment of CKD and ESKD imposes substantial societal costs with the highest medical costs for kidney replacement therapies; therefore, establishment of novel therapeutic approaches is desired. While adult kidneys possess an intrinsic capacity to self-repair after injury, the process of nephrogenesis, the formation of new nephrons, is limited to the period of embryonic development in humans. Human induced pluripotent stem cells (hiPSCs), by virtue of their unlimited self-renewal and ability to generate cells of all three germ layers of the embryo, are ideally suited for generation of functional human kidney cells and tissues. hiPSCs can be generated from patients with kidney disease, providing an immunocompatible source for kidney regenerative therapies. Significant advances have been made within the past decade that draw upon our knowledge of kidney development to differentiate hiPSCs into cells of the kidney lineage. By recapitulating metanephric kidney development in vitro, we generated nephron progenitor cells (NPCs) with ~90% purity within 8-9 days of differentiation without additional subpopulation selection during the directed differentiation. hiPSC-derived NPCs possess the developmental potential of their in vivo counterparts, forming kidney organoids which contain multiple nephron structures. Although advances in generation of kidney tissue from hiPSCs are significant, there remain many challenges to develop functional bioengineered kidneys as a novel source for kidney replacement therapies. The major limitation of the current form of kidney organoids is lack of perfusable vasculature to produce primitive urine via blood filtration by endothelia and podocytes of the glomerulus. Vascularization is also critical to generate and maintain large kidney tissue in vitro via oxygen supplies. While current approaches such as organ-on-chip technologies focus on simulating vasculature-tubules or -podocytes interaction by seeding each cell type in each channel, our paradigm shift approach utilizes intrinsic vasculogenic capacity to vascularize whole kidney organoids where multiple cellular communications are recreated in vitro. We will identify critical cues to stimulate endogenous vasculogenesis in kidney organoids to generate nephron capillary networks including glomerular capillary loops, an essential structure to produce primitive urine via blood filtration. Further, we will reproduce artery-nephron capillaries-vein connection by simulating native kidney vascular development via angiogenesis of renal artery. Our proposed study will enable primitive urine production in vitro and significantly advance the stem cell technology toward generation of functional bioengineered organs with identification of critical cues for vascularization in vitro.